Effect Of TGF-β1 Antsense Oligodeoxynucleotides To The Proliferation And Differentiation Of Human Rhabdomyosarcoma Cells

Rhabdomyosarcomas (RMS), the most common soft tissue sarcomas in childhood, although expressing some myogenic regulatory factors( MRFs), such as MyoDl, RMS cells undergoes very limited muscle differentiation and fail into terminal skeletal muscle differentiation. Some research reported that RMS cells can express growth factors which are likely to be involved in the growth and differentiation of tumor cells. Transforming growth factor ( TGF)-p is a multifunctional cytokine that regulate growth and differentiation of various cell types. TGF β1 is a potent growth inhibitor, with tumor-suppressing activity ,cancers are often refractile to this growth inhibition because of genetic loss of TGF- β1 signaling components. However, TGFβ1, a inhibitor of muscle differentiation , as well as an autocrine product of RMS cells, maybe regulate the tumor cells growth and differentiation by some way which has not yet been investigated. To study the role of TGF- β1 in the human rhabdomyosarcoma cell line, RD, we have made an attempt to establish a framework for the expression of components of TGFβ1/Smad signaling pathway in RD cells compared with the normalmyoblasts by RT-PCR and Western blot. We have shown that RD cells displayed higher expression of TGF-PU TGFpR I, TGFpR IK Smad2and Smad4 at both the mRNA and protein levels than myoblasts, and suggest that the RD cells line is a suitable model to study the role of TGF- ￡ 1 signalling in RMS. To date, little is known of TGF-13 1 action in RMS cells. In the present study, we use this model system to investigate whether the autocrine TGF- P 1 loop is responsible for cell growth and differentiation in RD cell.Antisense oligodeoxyribonucletide designed to inhibit target gene expression would be useful in assessing possible roles of some growth factors in cellular regulation. Indeed, antisense oligodeoxyribonucletide have been used to target TGF- 3 1 in attempt to evaluate its involvement in the formation and development of tumor. However, the effects of oligodeoxyribonucletide on TGF- P 1 production was not determined, this is very important, as will be demonstrated in our study, because oligodeoxyribonucletide treatment can lead to non-specific biological effects. In this research, we designed different oligodeoxyribonucletide to target TGF- P 1 mRNA, and choose one of best antisense oligodeoxyribonucletide, which specifically inhibits the production of TGF- & 1, transfer to the human rhabdomyosarcoma cell line, RD, to investigate the involvement of TGF- P 1 in cell growth and differentiation. The summarized results as follows:1. Immunohistochemical technique(S-P) and RT-PCR were used todetect the expression of TGF-pK TGFpR I, TGFPRIK Smad2 and Smad4 inrhabdomyosarcoma and normal skeletal muscles. Overexpression of TGFpi,Smad2, and Smad4 in RMS were 58.6% (41/70) , 70% (49/70) and 75.7%(53/70) respectively. The highexpression of above proteins in normalskeletal muscles were 28% (7/25), 24% (6/25) and 28% (7/25) with significant difference ( P <0.01) . Expression of TpR I , TpR II were detected in most of RMS and all of normal skeletal muscles without significant difference ( PX).O1) The mRNA expression of TGF(31, TpR I , TPR II, Smad2 and Smad4 in RD cell line is higher than that of the myoblasts. There is no significant relationship among the expression of above proteins, histological grade and prognosis of tumor (PO.05) except for expression of TGFpl protein in grade I and II. There exists an autocrine pathway in RMS, suggest that TGF- 1 may play a complex role in development and progression of rhabdomyosarcoma.2. To observe the biological properties of primary human embryonic skeletal myoblasts cultured in different medium with RT-PCR, flow cytometry (FCM) and rate of myotube formation (RMF), and to investigate the effect of transforming growth factor- P 1 (TGF- P 1) to the proliferation and differentiation of myoblast. Results showed that the primary myoblasts cultured in DM (DMEM supplemented with 3% fetal bovine serum), in contrast with the control group in GM (DMEM supplemented with 10% fetal bovine serum), increased the rate of myotube formation (RMF), more myofilament formed and more myoblasts exited S phase. The expression of Myogenin mRNA was increased obviously than that of the control group, the change of MyoDl and Myf5 mRNA was not as well as Myogenin, These data indicate that different culture medium induce diverse biological function of myoblasts, and provide a means of study role of TGFpi in RMS cell and myoblast. With TGF- P 1 (^2ng/ml) cultured for 6 days, all differentiation index of myoblasts above is decreased ,and more cells of experimental groupentered the phase of DNA duplication, however, with TGF- P 1 (^ lng/ml) cultured, exert little effect on growth and differentiation of myoblast. All of theses suggested that weather or not TGF- 0 lin RMS exert growth inhibitory effect in a concentration-dependent manner.3. To establish a credible way of transfer TGF-pi antisense oligonucleotide (TGF- P 1ASON) to RD cells, and provide a possible method of research in thepathogenic role of TGF-01 on rhabdomyosarcoma. TGF-piASON were synthesized, makered by 6-carboxy-iluorescein ( 6-FAM) and combined to LipoGenTM to prepare Lip- ASON complex. To analysis the property, dose of complex, and transfer time, results showed that the transfer efficiency is close to relation with all of these. The mean fluorescent index by FCM combined the transfer rates by manual counting can more objectively inspect transfer efficiency. Proper dose of Lip- ASON complex can improve transfer efficiency to maximum and with least of poison. The TGF-{$1 mRNA and protein of transferred RD cells are investigated by RT-PCR and ELISA, showed that TGF-fll ASON2 (3* gggggtacggcgggagccs' ) maybe is the best antisense to TGF-B1 mRNA, provide a means of research the role of TGF-61 in the RMS in the next part.4. To investigate the effect of TGF-61 ASON on the growth and differentiation of RD cells by MTT, FCM, RT-PCR, Immunofluorescent and electron microscope, and to study the mechanism of it by Western blot combined with RT-PCR to explore changes of Smads and MRFs. Results showed that TGF-81ASON could induce RD cells exit cell cycle, and higher protein expression of Myosin and a-Sarcomeric Actin than that of control group. It suggested that TGF- ￡ 1ASON could inhibit growth of RD cells andinduce differentiation of RD cell. Using RT-PCR with sequence-specific primers and Western blot with specific antibody, we have shown that TGF- P 1 ASON could reduce the expression of Smad2 and Smad4, and increase the expression of some MRFs, such as myogenin and MyoDl. These study suggested that TGF- P 1ASON broken off TGF- P 1 signaling pathway, stopped uniting Smads with MRFs, and induced RMS cells' cytoskeletons protein expression more, such as Myosin and a-Sarcomeric Actin.In summary, there is TGF- P 1 autocrine loop in RD cells, on the use of ASON transfer ,showed that TGF- 3 1ASON could inhibit growth of RD cells and induce differentiation of RD cell. In this study, we provide an application of ASON transfer to the research of growth factors of cells, conformed that TGF-J31 inhibit muscle differentiation of RD cells, and the result maybe from Smads cooperating with MRFs to inhibit muscle transcription of cytoskeletons gene.